**1. Introduction**

Infertility affects up to 15% of couples attempting to conceive globally, with a male factor implicated in up to 50% of cases [1]. While the precise etiology remains unclear in many of these cases, azoospermia, or the lack of sperm in the ejaculate, occurs in 1% of all males and 10–15% of infertile males and is often considered the most severe phenotype of male infertility classified as either obstructive azoospermia (30–40% of azoospermia cases) or non-obstructive azoospermia (NOA) (60–70% of azoospermia cases) [2–6]. NOA remains a particularly challenging condition to treat as the majority of cases are idiopathic, with only a subset attributable to an identifiable genetic (i.e., Klinefelter Syndrome, Y-chromosome microdeletion or mutations in individual genes) or acquired (i.e., chemotherapy, radiation, cryptorchidism/prior orchiopexy or malignancy) condition [7,8]. NOA men have spermatogenic failure with a range of histopathologic changes that include hypospermatogenesis, maturation arrest, and Sertoli cell only syndrome [7]. Currently, NOA men require surgical retrieval of sperm with assisted reproductive technology to father children. This review highlights milestones in the evolution of surgical sperm retrieval methods, summarizes predictors of sperm retrieval success, evaluates the data of conventional microdissection testicular sperm extraction (cTESE) versus microdissection testicular sperm extraction (mTESE), discusses tips for optimizing sperm retrieval, and comments on the future of sperm retrieval in men with NOA.

**Citation:** Punjani, N.; Kang, C.; Schlegel, P.N. Two Decades from the Introduction of Microdissection Testicular Sperm Extraction: How This Surgical Technique Has Improved the Management of NOA. *J. Clin. Med.* **2021**, *10*, 1374. https:// doi.org/10.3390/jcm10071374

Academic Editors: Giovanni M. Colpi and Ettore Caroppo

Received: 4 March 2021 Accepted: 26 March 2021 Published: 29 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. History of Surgical Sperm Retrieval**

While NOA men currently rely on surgical sperm retrieval with assisted reproductive technology to father biological children, these men were historically relegated to using adoption or use of donor sperm to have a family [9]. The first successful in vitro fertilization (IVF), the process by which a sperm and oocyte are fertilized outside of the body and then later implanted, was performed in 1978 utilizing ejaculated sperm from a fertile man, and resulted in the birth of Louise Brown (Figure 1) [10]. Sperm was surgically retrieved for the use of IVF for the first time in the 1980s, utilizing motile sperm from the epididymis of man with obstructive azoospermia [11]. Intracytoplasmic sperm injection (ICSI), a process where only a single sperm is injected into an oocyte using a micropipette, was then introduced in 1992, thereby potentially providing an opportunity for men with severe spermatogenic dysfunction (i.e., men with NOA) to father children [12]. The first description of testicular sperm for use in assisted reproduction occurred in 1993 [13]. Successful fertilization, embryo development, implantation and pregnancy was considered to be an unanticipated result by some reproductive experts given the expected need for additional maturation by sperm that was known to occur during epididymal transit [14].

**Figure 1.** Timeline of the history of surgical sperm retrieval prior to the discovery of mTESE. IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; NOA: non-obstructive azoospermia; mTESE: microdissection testicular sperm extraction.

It was not until 1995 that testicular sperm extraction (TESE), an open surgical procedure to directly extract testicular tissue, was performed on a man with NOA and testicular sperm utilized for successful IVF-ICSI [15]. This success was revolutionary for the treatment of men with NOA, but certain challenges remained given the sporadic, almost anecdotal, success of initial efforts to treat men with NOA who were previously considered to be sterile [16]. Based on observations that limited sperm was retrieved during simple biopsy, it was originally thought that multiple testis biopsies would increase the chance of retrieval as sperm production was believed to occur in isolated areas in the testis of men with NOA and spermatogenic failure [14]. In reality, multiple open biopsies resulted in removal of large quantities of testicular tissue and created a new risk, namely of harm to the blood supply of the testis from multiple incisions on the tunica albuginea. These incisions threatened to divide the vessels under the surface of the tunica vaginalis, with a potential risk of testis devascularization [17]. Percutaneous needle aspiration of the testis provided a minimally invasive alternative to sperm retrieval in NOA, but the lower sperm yield often did not provide enough sperm to inject all oocytes during an attempted ICSI attempt [18]. Given the risk of vascular injury, an approach to widely opening the testis and identifying individual tubules with the aid of an operating microscope was initiated. With this additional magnification, differences in seminiferous tubules could be

visualized and appreciated, in particular differences shown to reflect potential focal sites of sperm production in an otherwise highly dysfunctional testis. Additional considerations of the intratesticular blood supply that runs parallel to seminiferous tubules within the testis allowed the development of microdissection testicular sperm extraction (mTESE) by Schlegel et al. in 1998 [19]. Additional studies have documented enhanced sperm retrieval rates with this technique, as well as the additional safety of mTESE, by eliminating the need for multiple biopsies or incisions of the tunica albuginea, reducing the impact to the testicular blood supply [20,21].

#### **3. Surgical Testicular Sperm Extraction**

TESE is the surgical removal of tissue from the testicle in order to retrieve sperm, and can be completed with or without a standard operating microscope. cTESE may be completed with local anesthetic or sedation, but is commonly completed under general anesthesia. A skin incision may be made in the scrotal midline or through a unilaterally transverse or longitudinal incision over the selected hemiscrotum. Dissection is carried through subcutaneous dartos tissue down towards the tunica vaginalis which is then opened to reveal the testis. If delivered, the testis should be examined to identify and avoid areas of prominent vascularity, commonly seen in the midline and lower poles of the testis. An ultra-sharp or ophthalmic blade is used to sharply enter the tunica albuginea. Gentle pressure is applied around the tunical incision to extrude a sample of seminiferous tubules (Figure 2A). The tubules are sharply excised with scissors and processed in spermappropriate media by mincing the tissue with scissors and passage through a 24-gauge angiocatheter. The specimen is then examined by a trained andrologist, under light or phase-contrast microscopy at 20× magnification, for the presence of sperm.

**Figure 2.** Graphical representation of a ( **A**) conventional testicular sperm extraction with or without an operating microscope and (**B**) microdissection testicular sperm extraction using an operating microscope.

mTESE is now considered the gold standard procedure for sperm retrieval in men with NOA [22]. Under general anesthesia, a dissection identical to cTESE is performed, and the testis is delivered through an opening in the parietal tunica vaginalis. After delivery of the testicle, adhesions on the visceral tunica vaginalis should be released to ensure optimal visualization and location of vasculature. The tunica vaginalis is then sharply incised with an ultra-sharp or ophthalmic blade, but bi-valved equatorially (Figure 2B). The tunical edges are secured with clamps to prevent avulsion of the tunica from the underlying tubules. The seminiferous tubules are then examined carefully and systematically prior to manipulation. Bipolar cautery should be used to limit postoperative bleeding as well as tissue damage. The tissue should be manipulated with care to avoid disruption of individual seminiferous tubules or the vessels which run parallel to the tubules in a radial pattern from the center to the periphery of the testicular parenchyma. Once an optimal tubule is identified, the tubule should be taken in its entirety, whenever possible. Optimal tubules (i.e., those likely to contain sperm) are generally larger and more opaque [20]. Once an adequate number of tubules are selected, processing should occur as previously described using sharp scissors and aspiration through a 24-gauge angiocatheter. This mechanical disruption of testicular tissue can allow detection of rare sperm within the tissue that may not be identifiable in a sample that has not been similarly processed. Note that sperm, when present, are inside the seminiferous tubules, so the tubules must be broken open to release sperm into the tissue suspension. As previously described, a limited part of this dispersed tissue specimen can then be examined by an experienced andrologist using 20× phase contrast microscopy on a simple slide with cover slip.

#### **4. Predictors of Sperm Retrieval in mTESE**

Although many studies have been conducted examining sperm retrieval rates in men with NOA undergoing mTESE, limited definitive predictors of sperm retrieval exist.
